Recombinant adeno-associated virus (rAAV) is a promising vector for human gene transfer. Grimm, D., and Kleinschmidt, J. A. (1999) Hum Gene Ther. 10: 2445-2450; High, K. A. (2001) Ann. N.Y. Acad. Sci. 953: 64-67; Pfeifer, A., and Verma, I. M. (2001) Ann. Rev. Genomics Hum. Genet. 2: 177-211. AAV is a member of the Dependovirus genus of the parvoviruses. AAV serotype 2 (AAV2) is composed of a single-strand DNA molecule of 4680 nucleotides encoding replication (rep) and encapsidation (cap) genes flanked by inverted terminal repeat (ITR) sequences. Berns, K. I. (1996) in Fields Virology (B. N. Fields et. al. Eds.), pp. 2173-2197. Lippincott-Raven Publishers, Philadelphia. The genome is packaged by three capsid proteins (VP1, VP2 and VP3), which are amino-terminal variants of the cap gene product. The resulting icosahedral virus particle has a diameter of ˜26 nm. A high resolution crystal structure of AAV2 has been reported. Xie, Q. et al. (2002) Proc. Natl. Acad. Sci. USA. 99: 10405-10410.
The solubility of purified AAV2 virus particles is limited, and aggregation of AAV2 particles has been described as a problem. Croyle, M. A. et al. (2001) Gene Therapy 8: 1281-1290; Huang, J. et al. (2000) Mol. Therapy 1: 5286; Wright, J. F. et al. (2003) Curr. Opin. Drug Disc. Dev. 6: 174-178; Xie, Q. et al. (2004) J. Virol. Methods 122: 17-27. In commonly used buffered-saline solutions, significant aggregation occurs at concentrations of 1013 particles/mL, and aggregation increases at higher concentrations. Huang and co-workers reported that AAV vectors undergo concentration-dependent aggregation. Huang, J. et al. (2000) Mol. Therapy 1: S286. Xie and coworkers (Xie, Q. et al. (2004) J. Virol. Methods 122: 17-27) similarly reported that at concentrations exceeding 0.1 mg/mL, AAV2 vectors require elevated concentrations of salt to prevent aggregation. Aggregation of AAV2 vectors occurs at particle concentrations exceeding 1013 particles/mL in commonly used neutral-buffered solutions such as phosphate- and Tris-buffered saline. This corresponds to a protein concentration of ˜0.06 mg/mL, and emphasizes the low solubility of AAV2 under these conditions. The effective vector concentration limit may be even lower for vectors purified using column chromatography techniques because excess empty capsids are co-purified and contribute to particle concentration.
Particle aggregation is a significant and not fully resolved issue for adenovirus vectors as well. Stability of a recently established adenovirus reference material (ARM) was recently reported. Adadevoh, K. et al. (2002) BioProcessing 1(2): 62-69. Aggregation of the reference material, formulated in 20 mM Tris, 25 mM NaCl, and 2.5% glycerol at pH 8.0, was assessed by dynamic light scattering, photon correlation spectroscopy and visual appearance. A variable level of vector aggregation following either freeze-thaw cycling or non-frozen storage was observed, resulting in restrictive protocols for the use of the ARM.
Aggregation can lead to losses during purification and inconsistencies in testing of purified vector preparations. The in vivo administration of AAV2 vectors to certain sites, such as the central nervous system, may require small volumes of highly concentrated vector, and the maximum achievable dose may be limited by low vector solubility.
Vector aggregation is also likely to influence biodistribution following in vivo administration, and cause adverse immune responses to vectors following their administration. As has been reported for proteins (Braun, A. et al. (1997) Pharm. Res. 14: 1472-1478), aggregation of vector may increase immunogenicity by targeting the vector to antigen presenting cells, and inducing enhanced immune responses to the capsid proteins and transgene product. The reports of immune responses to AAV vectors in pre-clinical (Chenuaud, P. et al. (2004) Blood 103: 3303-3304; Flotte, T. R. (2004) Human Gene Ther. 15: 716-717; Gao, G. et al. (2004) Blood 103: 3300-3302) and clinical (High, K. A. et al. (2004) Blood 104: 121a) studies illustrate the need to address all factors that may contribute to vector immunogenicity.
Testing protocols to characterize purified vectors are also likely to be affected by vector aggregation. Determination of the infectivity titer of vector was reported to be highly sensitive to vector aggregation. Zhen, Z. et al. (2004) Human Gene Ther. 15: 709-715. An important concern is that vector aggregates may have deleterious consequences following their in vivo administration because their transduction efficiency, biodistribution and immunogenicity may differ from monomeric particles. For example, intravascular delivery of AAV vectors to hepatocytes requires that the vectors pass through the fenestrated endothelial cell lining of hepatic sinusoids. These fenestrations have a radius ranging from 50 to 150 nm (Meijer, K. D. F., and Molema, G. (1995) Sem. Liver Dis. 15: 206) that is predicted to allow the passage of monomeric AAV vectors (diameter ˜26 nm), but prevent the passage of larger vector aggregates. In biodistribution studies in mice, aggregated AAV2 vectors labeled with the fluorescent molecule Cy3 were sequestered in liver macrophages following vascular delivery. Huang, J. et al. (2000) Mol. Therapy 1: S286.
Formulation development for virus-based gene transfer vectors is a relatively recent area of investigation, and only a few studies have been reported describing systematic efforts to optimize AAV vector formulation and stability. Croyle, M. A. et al. (2001) Gene Therapy 8: 1281-1290; Wright, J. F. et al. (2003) Curr. Opin. Drug Disc. Dev. 6: 174-178; Xie, Q. et al. (2004) J. Virol. Methods 122: 17-27. Defining formulations compatible with pre-clinical and clinical applications that minimize changes in vector preparations is an important requirement to achieve consistently high vector safety and functional characteristics. As is well established for protein therapeutics (Chen, B. et al. (1994) J. Pharm. Sci. 83: 1657-1661; Shire, S. J. et al. (2004) J. Pharm. Sci. 93: 1390-1402; Wang, W. (1999) Int. J. Pharm. 185: 129-188; Won, C. M. et al. (1998) Int. J. Pharm. 167: 25-36), an important aspect of vector stability is solubility during preparation and storage, and vector aggregation is a problem that needs to be fully addressed. Vector aggregation leads to losses during vector purification, and while aggregates can be removed by filtration, the loss in yield results in higher costs and capacity limitations when producing vector for pre-clinical and clinical studies. Even after filtration to remove aggregates, new aggregates can form in concentrated preparations of AAV2 vector in buffered-saline solutions.
The need exists for improved formulations and methods for purification and storage of AAV vectors, such as rAAV2, that prevent aggregation of virus particles.